Capping Protein (CP) is a ubiquitously-expressed, 62 kDa heterodimer that binds the barbed end of the actin filament with very high affinity (0.1 nM) to prevent further monomer addition and loss. CARMIL is a multi-domain protein, present from protozoa to mammals, that binds CP and appears to be important for normal actin dynamics in vivo. CARMILs CP binding site resides in its CAH3 domain, a small domain located at or near the proteins C-terminus. CAH3 binds CP with 1 nM affinity, resulting in a complex with weak barbed end capping activity (30-200 nM). Moreover, solution assays and single-molecule imaging show that CAH3 can bind to CP already present on the barbed end, causing a 300-fold increase in CPs rate of dissociation from the end i.e. uncapping. Here we used nuclear magnetic resonance (NMR) and intermolecular paramagnetic relaxation enhancement experiments to define the molecular interaction between the minimal CAH3 domain (CAH3a/b) of mouse CARMIL-1 (mCARMIL-1) and CP. Specifically, we show that the CAH3a sub-domain is required for CAH3s high affinity interaction with CP. This interaction consists largely of electrostatic interactions between the highly basic CAH3a sub-domain and a complementary acidic groove on CP that lies opposite its actin-binding surface. This CAH3a: CP interaction serves to orient the CAH3b sub-domain, which we show to also be required for potent anti-CP activity, directly adjacent to the basic patch on CP, shown previously to be required for CPs association to and high affinity interaction with the barbed end. The importance of specific residue interactions between CP and CAH3a/b were confirmed by site-directed mutagenesis of both proteins. Together, these results offer a mechanistic explanation for the barbed end uncapping activity of CARMIL, and they identify the basic patch on CP as a crucial regulatory site. Capping protein (CP) is a ubiquitously-expressed, heterodimeric, 62 kDa protein that binds the barbed end of the actin filament with high affinity to block further filament elongation. V-1/myotrophin is a 13 kDa, ankyrin repeat-containing protein that binds CP tightly, sequestering it in a totally inactive complex in vitro. Here we elucidate the molecular interaction between CP and V-1 by nuclear magnetic resonance (NMR). Specifically, chemical shift mapping and intermolecular paramagnetic relaxation enhancement experiments reveal that the ankyrin loops of V-1, which are essential for V-1: CP interaction, bind the basic patch near the joint of CPs &#61537;tentacle shown previously to drive most of CPs association with and affinity for the barbed end. Consistently, site-directed mutagenesis of CP shows that V-1 and the strong electrostatic binding site for CP on the barbed end compete for this basic patch on CP. These results can explain how V-1 inactivates barbed end capping by CP and why V-1 is incapable of uncapping CP-capped actin filaments, the two signature biochemical activities of V-1.